This program investigates regulatory phenomenon involving transfer RNAs, tRNAs, within the context of a differentiating system, Bacillus subtilis. The long-term objectives are to relate these basic phenomena to broader questions on the physiology, evolution, and genetic regulation of Gram-positive bacteria, some of which cause human disease, and on the involvement of alterations in tRNAs, which accompany virus infections, differentiation, and neoplasia in processes that may have regulatory significance. The specific aims of this project are to examine the organization and expression of tRNA genes in B. subtilis, stressing regulation of transcription of the tRNA genes during development, and to investigate processing of the precursor-tRNAs. Experiments will be done to identify promoters within the 21-tRNA gene cluster that we have cloned and sequenced. Expression of these tRNAs will be examined in B. subtilis to elucidate mechanisms affecting the transcription of stable RNA species. Expression in vivo will be determined by an elevation in the levels of tRNAs; promoter regions will be located by S1-type mapping. Promoter regions will also be sought within other tRNA gene clusters. The activity of these tRNA promoters will be studied in promoter-probe plasmids examined in B. subtilis at various growth stages. In addition, studies on the processing of the 5'-termini of B. subtilis tRNAs will be continued. In particular, synthetic tRNA genes with alterations in the 3'-terminal CCA sequence and the aminoacyl stem will be used to generate natural or "mutant" pre-tRNAs as substrates for processing. Processing of these pre-tRNAs will be done in in vitro assays with B. subtilis RNase P and its catalytically active moiety, P-RNA.